DNA origami folding and purification
The DNA origami constructions (6HB, 24HB, 60HB, 13HR and nanocapsule) have been folded in a one-pot response by steadily reducing the temperature utilizing a Proflex 3 × 32-well PCR system (Thermo Fisher). The scaffold strands (p7249, p8064 and p7560 variants of single-stranded M13mp18) have been bought from Tilibit Nanosystems and the staple strands from Built-in DNA Applied sciences. To make sure excessive folding yields of DNA origami, structure-specific optimized situations relating to each the annealing procedures and the buffer selection (‘folding buffer’, FOB) are used (Supplementary Be aware 20).
Buffer change for DNA origami
The purified DNA origami constructions have been transferred into 6.5 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer supplemented with 2 mM NaOH (HEPES-NaOH, pH 6.5) earlier than complexation with CCMV CPs. The buffer change was carried out by spin-filtration59 utilizing 100 kDa molecular weight cut-off (MWCO) centrifugal filters (Amicon), which have been washed earlier than use by centrifuging with 400 μl of HEPES-NaOH buffer for five min at 14,000g. Subsequently, equal volumes of DNA origami answer and HEPES-NaOH buffer have been added into the filter machine and the centrifugation was continued for 10 min at 6,000g. A quantity of HEPES-NaOH equal to 2.09× the preliminary quantity of origami answer was then added, and the centrifugation step was repeated. The pattern was collected by inverting the filter and centrifuging for two.5 min at 1,000g.
Isolation of CCMV CPs
The CPs have been remoted from intact CCMV (for virus preparation, see Supplementary Be aware 21). Briefly, the virus particles have been dialysed in a single day in opposition to 50 mM Tris–HCl, 500 mM CaCl2 buffer, pH 7.5 supplemented with 1 mM dithiothreitol (DTT) utilizing Slize-A-Lyzer Mini Dialysis cups (3.5 kDa MWCO, Thermo Scientific). The RNA was pelleted in a centrifugation step at 4 °C utilizing 21,100g for six h, and the recovered supernatant was dialysed in a single day in opposition to ‘clear buffer’ that comprises 50 mM Tris–HCl, 150 mM NaCl at pH 7.5 supplemented with 1 mM DTT (tailored from ref. 60). The focus of the proteins was decided primarily based on their absorbance at 280 nm (extinction coefficient, 23,590 M−1 cm−1) utilizing a BioTek Eon Microplate Spectrophotometer (2 μl pattern, Take3 plate).
AGE
AGE was used to check the binding interplay between the proteins and the origami constructions by monitoring the shift in electrophoretic mobility. Moreover, the intactness of the origami constructions after folding and purification, and through DNase I digestion, was analysed by gel electrophoresis. To this finish, samples (volumes starting from 10 to 32 μl) supplemented with 6× gel loading dye (40% sucrose with out dye for samples from digestion research) have been run in a 2% (w/v) agarose gel (1 × Tris–acetate–ethylenediaminetetraacetic acid (TAE) buffer, 11 mM MgCl2) for 45 min at 90 V in 1 × TAE buffer supplemented with 11 mM MgCl2. For staining, ethidium bromide (EtBr) at a closing focus of 0.46 μg ml−1 was used and the DNA was visualized underneath ultraviolet mild utilizing a GelDoc XR+ system (Bio-Rad).
Complexation of DNA origami and CCMV CPs
The complexation between CPs and DNA origami was carried out at a closing origami focus of 4 nM (10 μl samples). The origami was added in a 1:1 quantity ratio to the protein answer that had been diluted within the ‘clear buffer’. Relying on the required protein extra, ε, which describes the molar ratio between CP to DNA origami, protein options starting from 0 to 60 μM (comparable to ε = 0–15k) have been ready. The NaCl focus was adjusted to 150 mM, leading to a complexation buffer containing 3.25 mM HEPES-NaOH, 25 mM Tris–HCl, 150 mM NaCl and 0.5 mM DTT. The complexation was carried out at 4 °C for at the very least 1 h and subsequently analysed utilizing AGE and TEM.
DNase I digestion assays
To review the safety impact of the CP coating in opposition to degradation of the origami constructions by DNase I, 2 μl of DNase I inventory (starting from 0 to 500 KU ml−1) was added to 16 μl of the pattern. Moreover, CaCl2 and MgCl2 concentrations have been adjusted, leading to a closing response quantity of 20 μl containing 3.2 nM DNA origami, 2.6 mM HEPES-NaOH, 20 mM Tris–HCl, 120 mM NaCl, 0.4 mM DTT, 1 mM CaCl2 and 5 mM MgCl2. The samples are incubated at 37 °C for 15 min (6HB) and 60 min (24HB). Earlier than analysing the result by AGE, samples complexed with CPs have been disassembled utilizing heparin sodium salt as a aggressive binding agent (closing concentrations of 1.5 μM for 6HB-2k and 24HB-2.5k and 82 μM for 6HB-10k and 24HB-10k; Supplementary Be aware 14).
RNA–DNA origami folding and purification
For the RNA–DNA hybrid origami (RNA-6HB), EGFP mRNA (CleanCap EGFP mRNA, TriLink Bio Applied sciences, L-7601) was used because the scaffold. In a one-pot response, the 996-nt-long mRNA scaffold was thermally annealed with 29 staple strands (bought from Built-in DNA Applied sciences, see Supplementary Be aware 22) into a brief 6HB construction utilizing a Proflex 3 × 32-well PCR system (Thermo Fisher). The construction is designed to include two scaffold crossovers and has a helical pitch of 11 bp per flip. For the folding response, the mRNA and the staples have been diluted into 1 × FOB containing 1 × TAE pH 8.4, 5 mM MgCl2 and 1 mM NaCl reaching closing concentrations of fifty nM and 500 nM, respectively. The response combination was incubated at 55 °C for 15 min61 and cooled down by inserting it on ice for at the very least 10 min earlier than storage at 4 °C. To validate the folding, 4 staple strands have been exchanged with staple strands containing a 3′ overhang (labelled with F, Supplementary Desk 2). A fluorophore-containing attachment strand (ATTO590, Built-in DNA Applied sciences), which was added to the folding combination in 10× extra per attachment web site, can then be built-in into the construction by hybridization with the staple overhangs.
The folded constructions have been purified from extra staple strands by spin-filtration. To this finish, the filter (100 kDa MWCO, Amicon) was washed with 400 μl of 1 × FOB by centrifugation at 14,000g for five min, adopted by two-times addition of 40 μl RNA-6HB along with 40 μl of 1 × FOB. After a centrifugation step at 6,000g for 10 min, 80 μl of 1 × FOB was added and the centrifugation continued (6,000g, 10 min). This washing step was repeated in whole thrice earlier than the pattern was recovered by inverting the filter right into a clear tube (1,000g, 2.5 min). The focus was decided by measuring the absorbance at 260 nm (extinction coefficient, 1.29 × 107 M−1 cm−1), and the profitable folding was decided by AGE (3.5 % (w/v) gels, visualization underneath ultraviolet mild (EtBr channel) and crimson mild (A647 channel), ChemiDoc MP system, Bio-Rad), AFM and TEM.
Complexation of RNA-6HB origami and CCMV CPs
For the complexation, purified RNA-6HB origami in 1 × FOB was blended with CCMV capsids in ‘clear buffer’ in a 1:1 ratio at a closing hybrid origami focus of seven.5 nM. This ends in a complexation buffer containing 45 mM Tris, 75.5 mM NaCl, 10 mM acetic acid, 2.5 mM MgCl2, 0.5 mM DTT and 0.5 mM EDTA. The samples have been incubated at 4 °C for at the very least 1 h earlier than evaluation with AGE and TEM.
Complexation of DNA origami and NoV CPs
NoVLPs have been ready as reported by Lampinen et al.62 and saved in 1 × phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4); nevertheless, right here SpyTag003 (ref. 63) has been fused to the C-terminus of the VP1 from the NoV pressure Hu/GII.4/Sydney/NSW0514/2012/AU. The particles have been high quality managed with dynamic mild scattering for particle formation, sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein purity and the residual dsDNA was measured. For the complexation with DNA origami, DNA origami was current within the pattern throughout each disassembly and reassembly of the VLPs. To this finish, the origami constructions have been transferred into deionized water utilizing spin-filtration (as described above). The DNA origami was blended with the NoVLPs at completely different concentrations in a 1:4 (v/v) ratio, leading to a closing origami focus of 6 nM (30 μl samples). The samples have been transferred into 3.5 kDa MWCO dialysis cups (Slize-A-Lyzer, Thermo Scientific) and dialysed in a single day at 4 °C in opposition to 50 mM Tris–HCl, pH 8.9. For reassembly, the samples have been, in a second step, dialysed in a single day at 4 °C in opposition to 100 mM sodium phosphate buffer, pH 6.0, equally as reported by White et al.64 The complexation throughout disassembly and meeting of the NoVLPs was analysed by AGE and TEM.
Complexation of DNA origami and SV40 CPs
The SV40 main CP VP1 (abcam, ab74565) was disassembled and reassembled (tailored from ref. 50) by dialysing the assembled VLPs in PBS in opposition to 20 mM Tris, 2 mM DTT, 5 mM EDTA and 50 mM NaCl, pH 8.9 for two h at 4 °C (3.5 kDa MWCO, Slize-A-Lyzer, Thermo Scientific), after which the EDTA focus was decreased by a further dialysis step at 4 °C for two h in opposition to 20 mM Tris, 2 mM DTT, 2 mM EDTA and 50 mM NaCl, pH 8.9. The focus was decided primarily based on the absorbance at 280 nm (VP1 extinction coefficient, 32,890 M−1 cm−1). The DNA origami was transferred into 100 mM HEPES buffer, pH 7.2, supplemented with 125 mM NaCl by spin-filtration (as described above). The proteins have been blended with the DNA origami in 1:1 (v/v) ratio to achieve closing concentrations of 0–20 μM and a couple of nM, respectively, and the samples have been incubated for twenty-four h at room temperature earlier than evaluation utilizing AGE and TEM.
Complexation of DNA origami and MPyV CPs
For the complexation of VP1 capsomers (for recombinant expression and purification, see Supplementary Be aware 23) and DNA origami, the origami constructions have been first transferred into 40 mM Tris buffer, pH 8.0, supplemented with 20 mM acetic acid, 2 mM EDTA and 12 mM MgCl2 utilizing spin-filtration (see above). Relying on the specified extra of proteins, ε, the capsomers have been diluted into ‘storage buffer’, containing 40 mM Tris, 200 mM NaCl, 1 mM EDTA, 5% (v/v) glycerol and 5 mM DTT, pH 8.0. For the complexation, VP1 capsomers have been diluted in a ratio of 1:20 within the origami answer, leading to a closing origami focus of 0.75 nM (30 μl samples) and a complexation buffer containing 40 mM Tris, 19 mM acetic acid, 1.95 mM EDTA, 11.4 mM MgCl2, 10 mM NaCl, 0.25% (v/v) glycerol and 0.25 mM DTT, pH 8. The complexation response was incubated at 4 °C in a single day earlier than evaluation with AGE and TEM.
AFM
A 20 μl droplet of 10 nM RNA-6HB origami answer (MgCl2 focus adjusted to 12.5 mM) was deposited on a freshly cleaved mica substrate (Electron Microscopy Sciences) for 1 min, adopted by three washing steps with 100 μl deionized water that was instantly blotted away. The pattern was dried underneath a gradual nitrogen stream and imaged instantly after pattern preparation. AFM photos have been acquired in air utilizing ScanAsyst in Air Mode along with ScanAsyst-Air probes (Bruker) on a Dimension Icon AFM (Bruker). Picture processing was carried out in NanoScope Evaluation v.1.90 (Bruker).
TEM
Plain DNA origami samples (4 nM) have been ready by incubation of a 3 μl droplet for 3 min on a plasma cleaned (20 s oxygen plasma flash, Gatan Solarus) Formvar carbon-coated copper grid (FCF400Cu, Electron Microscopy Sciences), which was subsequently blotted in opposition to filter paper and damaging stained. For CCMV-CP-complexed samples (4 nM DNA origami), a 3 μl droplet was deposited on the grid for 1.5 min. After blotting in opposition to filter paper, the grid was immersed in a ten μl droplet of complexation buffer (3.25 mM HEPES-NaOH, 25 mM Tris–HCl, 150 mM NaCl, 0.5 mM DTT) for five s. For samples with DNA origami concentrations ≤2 nM (for instance, complexation with SV40, MPyV), and for samples containing RNA-6HB (7.5 nM origami focus), the droplet measurement was elevated to five μl and the incubation time prolonged to five min. Destructive staining65 was carried out by first immersing the grid in a 5 μl droplet of aqueous 2% (w/v) uranyl formate answer (supplemented with 25 mM NaOH for pH adjustment), which was instantly blotted away. This step was adopted by an immersion in a 20 μl droplet, which was incubated on the grid for 45 s. After the ultimate blotting step, the samples have been left to dry for at the very least 20 min earlier than imaging was carried out on a FEI Tecnai 12 Bio-Twin microscope at an acceleration voltage of 120 V.
Cryo-EM
The samples for cryo-EM have been ready utilizing a vitrification equipment (Vitrobot, Thermo Fisher Scientific). The origami concentrations within the complexed samples have been 90 nM for 6HB-2k, 84 nM for 24HB-2.5k, 18 nM for 6HB-10k and 21 nM for 24HB-10k, leading to whole CP concentrations of 180 μM and 210 μM for complexed 6HB and 24HB samples, respectively. A 3 μl aliquot of the complexed origami pattern was deposited on a plasma-cleaned (50 s, Harrick Plasma PDC-002-EC instrument) holey carbon-coated grid (copper 200 mesh R1.2/1.3, Quantifoil). After a 1 min incubation, extra liquid was blotted for 10 s at 100% relative humidity and 6 °C, adopted by plunging the grid into liquid ethane. The grids have been saved in liquid nitrogen. Information have been collected at liquid nitrogen temperature in a Talos Arctica transmission electron microscope (Thermo Fisher Scientific) operated at 200 kV, utilizing a Falcon III direct electron detector (Thermo Fisher Scientific). A magnification of 150,000× was used, leading to a calibrated pixel measurement of 0.96 Å. The information assortment parameters are listed in Supplementary Desk 4 (Supplementary Be aware 24).
Single-particle reconstruction
Cryo-EM knowledge have been processed utilizing CryoSPARC 3.3.2 (Structura Biotechnology) except said in any other case. Distinction switch perform parameters have been estimated utilizing CTFFIND4 (ref. 66). Segments alongside filaments have been outlined utilizing the Filament Tracer perform. Helical symmetry parameters have been estimated initially from 2D class averages utilizing Python-based Helix Indexer67. The construction and helical symmetry parameters have been refined utilizing Helix Refine perform and non-uniform refinement on motion-corrected helix segments. To find out the helical symmetry parameters of the 6HB-10k outer layer, a second 2D classification run was carried out after subtracting the contribution of the interior layer utilizing the Particle Subtraction perform. The Helix Refine was run on the subset of particles that confirmed a transparent second layer, utilizing the decided symmetry parameters as preliminary estimates. Reconstructions have been sharpened by making use of an advert hoc B-factor of −300 Å2. The reconstructions have been averaged in actual area by imposing the helical symmetry parameters on the central, most ordered a part of the map (50% of the quantity) in Bsoft68.
For modelling the construction of the capsomer, CP monomer (PDB:1cwp) was fitted within the 6HB-2k reconstruction within the six positions of the hexamer as inflexible our bodies in UCSF ChimeraX 1.3 (ref. 69). The atomic mannequin was refined in opposition to the density utilizing ISOLDE 1.3 (ref. 70) and Phenix 1.19 (ref. 71). To create atomic representations of the filaments, symmetry copies of the hexamer have been created in ChimeraX. To visualise the location of CP hexamers and pentamers within the cap, the caps of the 6HB-2k filament have been manually picked within the micrographs. The cap construction was refined utilizing the Helix Refine perform omitting symmetrization, as this allowed limiting the lean angle of the caps near aspect views. Reconstruction of the cap was filtered to its native decision utilizing Native Filter. The hexamer atomic mannequin and beforehand decided pentamer construction (extracted from PDB:1cwp after making use of icosahedral symmetry) have been fitted as inflexible our bodies in ChimeraX 1.3. Information-processing parameters are given in Supplementary Desk 4. Mannequin refinement and validation parameters are proven in Supplementary Desk 5.
SAXS
The samples for SAXS have been ready at origami concentrations of 165 nM (6HB, comparable to a disassembled CP focus of 330 μM) and 180 nM (24HB, comparable to a disassembled CP focus of 450 μM) and sealed inside a 1.5-mm-diameter glass capillary. The measurements have been carried out utilizing a Xenocs Xeuss 3.0C machine outfitted with a GeniX 3D copper microfocus supply (wavelength λ = 1.542 Å) and an EIGER2 R 1M hybrid pixel detector at a pattern–detector distance of 1,100 mm. Information acquisition was carried out for 3 × 3 h per pattern. To acquire the 1D SAXS knowledge, the 2D scattering knowledge have been azimuthally averaged. The magnitude of the scattering vector q is given by (q,=,4uppi sin theta /lambda) with 2θ being the scattering angle. Information remedy included averaging of the triplicate 2D knowledge of every pattern, background subtraction from the complexation buffer (3.25 mM HEPES-NaOH, 25 mM Tris–HCl, 150 mM NaCl, 0.5 mM DTT) and a type issue was fitted to a cylinder (6HB, 24HB), spheres (T = 3 icosahedral CPs assemblies) and a core–shell cylinder (6HB-2k, 24HB-2.5k) utilizing SasView software program. A Debye–Anderson–Brumberger mannequin was added to account for the background.
